Vascular calcification contributes significantly to cardiovascular morbidity and mortality. It develops in atherosclerotic lesions in a process closely resembling osteogenesis. Paradoxically, it is associated, age independently, with osteoporosis. The mechanism is unknown. We previously showed that vascular cell calcification is, in fact, osteogenic differentiation, ultimately leading to bone tissue formation. The guiding hypothesis of this proposal is that some critical regulator(s) of skeletal bone metabolism also regulate, in a reciprocal manner, bone formation in the artery wall. Two of the major regulators are RANKL, a TNF-alpha superfamily member, and OPG, its soluble decoy receptor. In bone, RANKL promotes bone resorption by osteoclasts, which are abundant in this tissue. In the vasculature, however, where osteoclasts are rare, RANKL treatment does not promote resorption, but, more importantly, it appears to actively promote calcification. One possible mechanism for active calcification by RANKL may lie in its known activation of NF-kB. In chondrocytes, it has been shown that NF-kB induces the potent osteogenic factor, BMP-2. We propose that this pathway may be responsible, in part, for vascular calcification and its paradoxical association with osteoporosis. Significant recent evidence supports this hypothesis: 1) RANKL is upregulated, and OPG is downregulated, in calcified arteries, 2) mice deficient in OPG develop vascular calcification, and 3) RANKL induces osteogenesis and mineralization in valvular myofibroblastic cells. In preliminary studies, we have found that in vitro vascular cell calcification is induced by RANKL and inhibited by OPG. We also found that atherogenic lipids and hyperlipidemia inhibit OPG expression, which would leave RANKL activity unopposed. This mechanism offers a unified explanation for the paradoxical effects of atherogenic factors on biomineralization in bone and in the artery wall. We hypothesize that RANKL stimulates, and OPG inhibits, vascular calcification and that atherogenic lipids increase RANKL activity. In Aim 1, we will test whether vascular calcification in OPG deficiency results from unopposed RANKL activity and upregulation of BMP-2. We will also test whether another OPG ligand, TRAIL, contributes to calcification by sequestering OPG. In Aim 2, we will test whether atherogenic lipid inhibition of OPG expression results in unopposed RANKL activity. As a corollary, we will also test whether OPG treatment prevents atherosclerotic calcification. In Aim 3, we will test the hypothesis that RANKL directly induces vascular calcification in vivo by generating transgenic mice with RANKL overexpression in the vasculature (SM22-rankl(tg) mice). We will use a vascular-specific promoter to avoid the confounding effects of a bone phenotype. These proposed studies will elucidate the roles of RANKL and OPG in vascular calcification and may define a new therapeutic paradigm for calcific vascular disease. Artery wall calcification contributes to cardiovascular disease, and it paradoxically occurs in conjunction with osteoporosis. Based on new supportive evidence, this research will test whether specific regulatory factors that promote loss of mineral from bone also promote gain of mineral in the artery wall.